Marek's Disease Virus (MDV) is a herpesvirus, which causes lymphoproliferative disease in chickens. Even after the introduction of vaccines against MDV, the infection still causes considerable losses in the poultry industry. MDV is divided into three serotypes, all of which establish latent infections. Serotype 1 includes oncogenic viruses, serotype 2 non-oncogenic viruses and serotype 3 includes the turkey herpesviruses (HVT) (Bülow et al (1976) Zentralblatt für Veterinarmedizin, 23B, 391-402).
Handberg et al (2001) Avian Pathology 30: 243-249 describe the use of serotype 1- and serotype 3-specific PCR for the detection of MDV in chickens. Tissue samples were taken from blood (buffy-coat cells), spleen, liver, skin, feather tips and ovaries.
CVI988 (Rispens) strain, a naturally-attenuated MDV serotype-1 (MDV-1), was introduced in the mid-1990s (Rispens et al., (1972) Avian Diseases, 16, 108-125; de Boer et al., (1986) Avian Diseases, 30, 276-283) and is to-date the most effective vaccine against MDV. However, even in light of vaccination programmes, MDV outbreaks continue to cause significant losses. These outbreaks can have several causes including: inhibition by maternal antibodies of vaccine virus replication (King et al., (1981) Avian Diseases, 25, 74-81; de Boer et al., (1986) supra); suppression of the immune response to vaccine by environmental stresses or co-infections with immunosuppressive pathogens; infection with a virulent MDV strain prior to establishment of full vaccinal immunity; infection of vaccinated and fully immunocompetent birds with a hypervirulent MDV strain (Witter et al., (2005) Avian Pathology, 34, 75-90); and, finally, administration of insufficient vaccine dose which fails to induce protective immunity.
As such it is important to be able to accurately measure MDV vaccine and challenge (virulent) virus individually, including measuring one or both, in the same individual chicken for both experimental and commercial reasons. Experimentally, it is very useful to be able to examine the interactions between vaccine and challenge virus, with a view to better understanding the mechanism of vaccinal protection and differences in efficiency between vaccines. Commercially, it is important to assist in identifying the causes of vaccine failures, such as administration of a sub-optimal vaccine dose (Landman & Verschuren, 2003), interference with vaccine virus replication by maternal antibodies (King et al., 1981; de Boer et al., 1986), and infection with hypervirulent MDV field strains (Witter et al., 2005).
A highly sensitive real-time quantitative PCR (q-PCR) assay for absolute quantitation of MDV-1 in chicken tissues has been developed (Baigent et al., 2005a) and used very successfully to investigate the kinetics of CVI988 vaccine virus replication in tissues of experimental and commercial chicks in the absence of challenge virus infection (Baigent et al., 2005b, 2006). However this method cannot distinguish between CVI988 and challenge virus. Other groups have developed real-time PCR assays to quantify MDV-2 (Renz et al., 2006) and MDV-3 (herpesvirus of turkeys) (Islam et al., 2006a). These serotype-specific assays can be used to quantify both vaccine and virulent challenge virus in individual chickens, where challenge virus (always MDV-1) is of a different serotype to vaccine virus (MDV-2 or -3) (Islam et al., 2006b).
However, the most widely used and effective MDV vaccine is a naturally-attenuated serotype-1 strain (CVI988, Rispens) (Rispens et al., 1972; de Boer et al., 1986) and, to date, real-time PCR to distinguish challenge virus from serotype-1 vaccine virus has not been possible, because sequence differences between them are very limited. A difference in the number of repeats in the 132-bp repeat region can be used to distinguish CVI988 from virulent strains using conventional PCR (Becker et al., 1992; Silva, 1992). However, the repetitive nature of this region precludes its use in quantitative PCR. Differences in the meq gene and the ICP4 gene are not consistent between CVI988 and virulent strains. A qPCR system has also been developed (Baigent, unpublished) to distinguish between BAC (Bacterial Artificial Chromosome) cloned MDV-1 and wild-type MDV-1 by targeting the BAC-specific sequence of the BAC cloned MDV-1 and US2 gene of the wild-type MDV-1, this can be used to distinguish between BAC cloned vaccine strains and wild-type virulent strains but only when such vaccine stains are BAC cloned. Importantly, such a method could not be used commercially, since no commercial MDV-1 vaccines are BAC cloned.
The pp38 gene shows a consistent single nucleotide difference between the CVI988 vaccine strain and virulent strains of MDV-1, however q-PCR primers are difficult to design which would be able to distinguish such a small difference.